A majority of the drugs and fine chemicals in use today are natural products or their derivatives. The source organisms (e.g., trees, marine invertebrates) of many of these natural products are neither amenable to the large-scale cultivation necessary to produce commercially viable quantities nor to genetic manipulation for increased production or derivatization of these compounds. Therefore, the natural products must be produced semi-synthetically from analogs or synthetically using conventional chemical syntheses. Furthermore, many natural products have complex structures, and, as a result, are currently uneconomical or impossible to synthesize. Such natural products must be either extracted from their native sources, such as trees, sponges, corals and marine microbes; or produced synthetically or semi-synthetically from more abundant precursors. Extraction of a natural product from a native source is limited by the availability of the native source; and synthetic or semi-synthetic production of natural products suffers from low yield. Such low yields and limited availability of the natural source can restrict the commercial and clinical use of such products. The biosynthesis of natural products in microbes could tap the unrealized commercial and therapeutic potential of these natural resources and yield less expensive and more widely available fine chemicals and pharmaceuticals. In many instances, however, the enzymes involved in production of clinically or industrially important compounds in a living organism are unknown.
Terpenes are examples of such natural products that are structurally complex and difficult or impossible to synthesize with currently available methods. Terpenes have enormous commercial, scientific, and public health potential. The development of therapeutic terpenes is of particular interest for cancer treatment. Examples of known or potential pharmaceutically important terpenes are taxol, artemisin, eleutherobin, and the sarcodictyins.
Taxol and its derivative Taxotere are two powerful anti-cancer diterpenes used to battle not only breast and lung cancers but also Kaposi's sarcoma. The success of the diterpene Taxol, which was isolated from the bark of the pacific yew tree, has validated the importance of terpene natural products as chemotherapeutics. Eleutherobin and sarcodictyins are potential anti-cancer compounds that share a eunicellane backbone structure and exhibit Taxoid-like modes of action. Eleutherobin was first isolated in 1995 from a soft coral (Eleutherobia sp. Alcyonacea Alcyoniidae), while the sarcodictyins were first isolated in 1987 from the Mediterranean stoloniferan coral Sarcodictyon roseum. 
Despite the development of total chemical syntheses, supply limitations still hamper efforts to bring eleutherobin and the sarcodictyins to the clinic. Currently available synthetic routes for production of eleutherobin and the sarcodictyins are far too costly to satisfy the needs of clinical trials and to meet downstream demand. However, these synthesis studies have demonstrated that eleutherobin and its precursors can be used as starting materials for the chemical synthesis of derivatives. Economical production of eleutherobin and the sarcodictyins or of a common structural component for use as a chemical synthon is needed to further develop these promising anticancer compounds. As an alternate source of supply, eleutherobin can be isolated from the aquarium coral Erythropodium caribaeorum; however, based upon the large amounts that would be required each year to meet market demand, the slow growth rates of soft coral make harvesting eleutherobin from its natural source impractical.
Every year numerous terpene-derived compounds with promising therapeutic properties are discovered and isolated from corals, sponges, microbes, and plants. The commercial development of these molecules can be limited by the trace quantities present in the natural sources. Therefore, there is a continuing need to develop methods of expressing the terpene biosynthetic genes in microbes, to enable scarce terpenes to be produced in the quantities required for clinical use. In spite of the progress in this field, most commercially relevant terpene synthases have not been cloned and the number of cloned terpene synthases falls far short of the number of identified terpenoid compounds. In addition, the lack of sequence identity among terpene synthases from different organisms and the low-throughput nature of current cloning methods preclude rapid screening, identification and expression of these genes. Furthermore, existing gene discovery methods are time and labor intensive and not amenable to the high-throughput cloning of terpene synthases or the generation of large gene libraries for combinatorial biosynthesis.
Current methods for identification of previously uncharacterized enzymes include (1) use of DNA encoding a known enzyme to identify genes with a similar nucleic acid sequence; (2) use of partial protein sequences from a known, purified enzyme to design degenerate probes for screening libraries; and (3) use of a similarity-based polymerase chain reaction (PCR) where highly conserved regions of the enzyme are used to design degenerate PCR primers and amplify a region of a nucleic acid that encodes an enzyme related to the known enzyme. Such methods rely on some subset of the following criteria: the ability to target conserved sequences with a nucleic acid probe or oligonucleotides, the ability to generate enriched cDNA libraries, the ability to purify the enzyme from tissue, or the ability to express a functional enzyme in a library host. Thus, there is a need in the art for additional methods for identifying new enzymes, where the methods avoid, or are at least less hampered by, such limitations.
The present invention addresses this need by providing a method for identification of new enzymes.